Biology subjects

Volpato, A.

Publications and source records attributed to Volpato, A..

2 recordsLinked to original sources

bioRxiv2025

All-Optical Strategies to Minimize Photo-Bleaching in Reversibly Switchable Fluorescent Proteins

Photo-bleaching is a general hurdle of fluorescence-based techniques that becomes even more severe in high- resolution microscopy that relies on prolonged, focused and complex illumination sequences. Strategies to reduce photo-bleaching require chemical modifications of the cell media, which often stave off physiological cellular conditions. Here, we outline an all-optical strategy to minimize photo-bleaching in reversibly switching fluorescent proteins (RSFPs), a class of probes used in several super-resolution and protein-multiplexing imaging techniques. By identifying the photobleaching pathways, we developed novel imaging schemes to increase the number of ON- OFF photo-switching cycles based on a designed modulation of the on-switching light or a co-irradiation with red- shifted light. By rationalizing the photo-cycle, we expand multiplexing strategies with RSFPs to high- spatiotemporal resolutions while maintaining the accuracy and recording longer time-lapse imaging of sub-cellular structures with both confocal microscopy and parallelized RESOLFT nanoscopy.

biophysics↗

bioRxiv2024

Structural diversity of Arc oligomers within the excitatory synapse

The Activity-Regulated Cytoskeleton-Associated protein (Arc), is pivotal to mediate plasticity responses in neuronal cells. In vitro studies suggest its ability to form high- and low-order oligomers, which are involved in neuronal trafficking. Despite its important functions, no direct observation of Arc oligomers in cells has been presented due to its highly regulated spatiotemporal expression, the small size of the structures, the lack of appropriate labelling strategies and the background associated to free diffusing cytosolic proteins. Here, we apply super resolution microscopy to observe Arc oligomeric states in cellular environment with focus on the excitatory synapse. In cells, we provide the first evidence of Arc high-order oligomers; we uncovered intermolecular interactions of Arc, its tendency to form liquid condensates and interaction with lipid bilayers. Arc high-order oligomers affect AMPA receptor surface levels. Together, our observations suggest a model by which Arc oligomerization mediates plasma membrane negative curvature favoring AMPA receptors endocytosis.

neuroscience↗